Processed tomato products and process for preparing the same

ABSTRACT

Processed tomato products which have an increased consistency as expressed by reduced Bostwick at certain insoluble solids level and Brix value.

FIELD OF THE INVENTION

The present invention relates to processed tomato products and a processfor preparing tomato products.

BACKGROUND OF THE INVENTION

In the industry of processing tomatoes to end products like sauces,ketchups, soups, toppings, etcetera usually two stages aredistinguished: primary processing and secondary processing. Primaryprocessing usually involves at least either hot- or cold breaking of thetomatoes and a concentration step. In the concentration step water isremoved from the tomato pulp, so as to obtain a thick paste. The waterremoval can be done by many ways, although evaporative removal of water(by heating) is the common method. The so-obtained thickened paste orpuree can be stored or directly further processed into a range offinished products such as tomato sauce for pasta, tomato ketchup,etcetera.

Such end products generally need a specific thickness to be valued asquality products (next to a good colour, flavour, etcetera). In order toachieve this, it is preferred that the product has (at a givenpercentage of soluble solids) a high consistency. Consistency in thetomato industry is often measured and expressed as Bostwick value. Inthe handbook “Tomato Production, Processing & Technology” (3^(rd) ed.)by W. A. Gould, CTI Publications, Timonium, Md., USA it is set out onpage 329, 330 how Bostwick measurements on tomato purees and pastes isusually performed in the tomato processing industry and tomato research.

In part, the thickness is determined by the amount of insoluble solidspresent per unit of tomato product. The insoluble solids are in partcellulose, pectins and other compounds that make up the structuralmatrix of the fruit. The amount of insolubles may vary per variety,season, growth stage, etcetera. The degree of concentration of tomatoproducts is usually expressed in degrees Brix and is an indication ofthe amount of soluble solids in a tomato (product). To exemplify this: atomato paste of 20 Brix is considered to be twice as much concentratedas a paste of 10 Brix of the same tomatoes.

Of course, in order to achieve thick products one can highly concentratethe tomato puree. The result will be a product with a high degree ofBrix, a firm consistency (expressed by low Bostwick value). However,this is costly, as many kilograms of tomatoes are needed to produce onekilogram of tomato product, and the evaporative concentration is also acost factor. Furthermore, flavour and colour can be adversely affectedby rigorous concentration, e.g. due to burning in the evaporators.

Many techniques have been developed to thicken paste without changingBrix value. Such methods include treating of the pectic substances withenzymes, adding thickeners, etcetera. These methods all have theirdisadvantages.

Hence, there is a need for tomato paste having a firm consistency at areasonable Brix value. Also, there is a need for processed tomatoproducts other than tomato paste (in the strict sense of the word) whichhave an increased consistency. Furthermore, such tomato paste shouldhave an acceptable flavour and colour for a tomato paste. Apart frompaste as described above, there is also a need for processed tomatoproducts containing pulp or dices of tomato products having a goodfirmness and improved pulpiness, and manufacturing of such pulp or dicesand processed tomato products containing such pulp or dices should beconvenient (e.g. with low losses/size reduction due to pulp and/or dicesbeing disintegrated or reduced in size due to attrition in theproduction chain.

Tomatoes having impaired ripening are reported, e.g. due to specificmutations known as alc, nor, rin and Nr.

E. Kopeliovitch et al, Euphytica 28, 99-104 (1979) disclose improvedstorage life for ripening inhibited mutants rin, nor, Nr. Pigmentationis also discussed.

E. Kopeliovitsch et al, J. Amer. Soc. Hort. Sci. 107(3), 361-364 (1982)disclose the effect of genes rin and nor on the flavour of raw tomatoes.It is mentioned that fruits homozygous in rin or nor are inferior toother fruits as to their fruity flavour.

E. C. Tichelaar et al, CSIRO Fd Res. Q, 38, 22-24 (1978) disclose tomatofruit ripening, and more in particular in the influence of the nor geneon that.

E. C. Tichelaar et al, HortScience, 13(5), 508-513 (1978) discloseenzyme levels, color, shelf life and other characteristics of homozygousand heterozygous Nr, rin and nor tomatoes.

R. W. Buescher et al, J. Food Science, 44(1), 190-192 (1979) disclosecharacteristics of processed tomato products of heterozygous nor fruit(nor hybrid with Heinz variety H1439).

S. Malis-Arad et al, J. Hort. Science, 58(1), 111-116 (1983) disclosemeasurement of pectic substances in rin and nor tomatoes.

K. Davies et al, J. Plant Physiol. 139, 140-145 (1991) discloseinfluence of salt stress on ripening of nor tomato fruit.

M. L. de Araujo et al, Euphytica 125, 215-226 disclose analysis ofhomozygous and heterozygous alc tomatoes in combination with color genesogc and hp. These fruit were generated with the view to achieving normalfruit color and extended shelf life.

M. Mutschler et al, J. Amer. Soc. Hort. Sci., 109(4), 504-507 (1984)disclose ripening and storage characteristics of the alc tomato.

G. E. Hobson, J. Sci. Food Agric. 31, 578-584 (1980) discloses theeffect of Nr and rin genes on the composition, enzyme content andpotential use of such tomatoes.

Although from the above references it seems a range of properties ofsuch ripening-inhibited tomatoes due to one or more genes of alc, rin,nor or Nr have been studied, commercial use was not reported, and infact discouraged.

SUMMARY OF THE INVENTION

It has now been found that the above objectives may be achieved (atleast in part) by a tomato paste having an increased consistency suchthat when measured at an insoluble solids interval of 2.5-3.6% at 12°Brix:(Bostwick value)<10.5−2.3822×(percentage of insoluble solids),  (1)when Bostwick is measured as defined in the reference given above.

Preferably, this is achieved by a tomato paste having an increasedconsistency such that when measured at an insoluble solids interval of2.5-3.6% at 12° Brix:(Bostwick value)<10.0−2.3822×(percentage of insoluble solids).  (2)

Most preferably, this is achieved by a tomato paste having an increasedconsistency such that when measured at an insoluble solids interval of2.5-3.6% at 12° Brix:(Bostwick value)<9.5−2.3822×(percentage of insoluble solids).  (3)

As is stated above, the tomato paste when measured should have aninsoluble solids level of 2.5-3.6%, and at 12° Brix. Pastes withdifferent levels of Brix are also part of the invention, but need to beconcentrated/diluted before measurement. In the above, the Bostwickvalue will suitably be above 0.1 at said Brix level.

Tomato paste is herein to be understood as a commercially-processed (orfactory-processed) tomato paste as it is known in the art of tomatoprocessing. Such tomato paste is the result of primary processingtomatoes (comminution/heating and concentrating by removal of water) asis done shortly after harvesting. A hot break process is preferred foroptimal consistency. The resulting product is a concentrated paste,which can be stored until further use, or can be sold. There arecommercial producers of such tomato paste (product). For comparison andmeasuring, such paste should be free of added thickeners, such asstarches or gums. Also, for comparison and measuring, the pastes shouldnot have been subjected to additional processing steps that may increasethe consistency, such as homogenisation treatment. Conventional,commercially available paste is free of such additional thickeners orprocess steps.

Although for measuring the Bostwick value at 12° Brix as set out abovethe tomato paste is a paste obtained with a hot break process, withoutadditional process steps or ingredients that influence the consistency,the invention may be applied to all sorts of tomato paste (hot and coldbreak), which do comprise additional thickeners or process steps thatinfluence the consistency.

Of a commercially available paste one can measure Bostwick, Brix, andinsoluble solids, and such numbers can give an indication of the qualityof the paste.

Tomato paste can be obtained by a hot break process (comminuting andheating to approx. 80° C.), optionally followed by a concentration stepto bring it to the required Brix value. Such concentration (i.e.water-removal) will usually be done by evaporation. The tomato pasteaccording to the invention does not contain gums, starches, or otherthickeners when measuring Bostwick and Brix value. Bostwick is usuallymeasured at 12° Brix. If the tomato paste has a too high Brix value,dilution with water to the required value of 12° may be applied.

The tomato paste according to the invention preferably is red (reddish),yellow (yellowish), orange (orangish), or pink (pinkish). Preferably,the paste according to the invention has a USDA color score at 8.5° Brixof 35-60. Dilution may be needed to measure at the required Brix value.

There are factories (“secondary processing”) that buy/use tomato pastefor preparing processed tomato products, such as pasta sauce, juice,ketchup, etcetera. Such processed tomato products may also be preparedfrom tomato paste, or from fresh tomatoes. Following this, apart from atomato paste with an increased consistency, there is also a desire forprocessed tomato products with a good consistency.

One factor which limits the consistency that can be obtained fromprocessed tomato products is the softening of fruit that takes place aspart of the ripening process. Aspects of fruit ripening such asdevelopment of colour and flavour give rise to desirable characteristicsin processed tomato products. It would be advantageous to combine thehigh consistency of unripe fruit with the colour and/or flavour of ripefruit in a processed tomato product.

A number of genes are involved in controlling the process of tomatofruit ripening. Mutations in such genes can lead to ripening-inhibitedfruit in which all aspects of the ripening process, such as softening,red colour formation, and flavour development are inhibited. If themutation is present in homozygous form, softening is minimised, anddevelopment of both colour and flavour is severely restricted. Examplesof gene mutations that result in ripening-inhibition in tomatoes include‘alcobaca’ (alc), ‘ripening-inhibited’ (rin), ‘non-ripening’ (nor), and‘Never ripe’ (Nr).

It has been found that both tomato paste and processed tomato productswith a good consistency can suitably be achieved if tomatoes homozygousin alc, rin, nor, or Nr are used to prepare a tomato paste or processedtomato product. Hence, the invention further pertains to a paste orproduct comprising tomatoes which are homozygous for alc, homozygous forrin, homozygous for nor, homozygous for Nr, heterozygous forcombinations of (at least) two of the alc, rin, nor or Nr genes, orcombinations thereof. Such tomatoes are herein after referred to as“tomatoes according to the invention”.

In a preferred aspect of the invention, the paste or product is preparedby using tomatoes according to the invention and which in additioncomprise color enhancing genes such as old gold crimson (ogc), highpigment (hp), dark green (dg), intense pigment (Ip), or other colorenhancing transgenic genes. Such tomatoes can not only be used to maketomato paste but can be used for a whole range of processed tomatoproducts. The term “processed tomato product” is herein to be understoodas to comprise any product that comprises tomatoes which are subjectedto processing steps (in any order) such as heating and breaking andoptionally concentrating and packing. Examples of processed tomatoproducts are: tomato pastes, tomato sauces, tomato juices, tomatoconcentrates, tomato passatas, salsa, barbecue sauce, pizza sauce,spaghetti sauce, tomato fritto, ketchup (catsup), soup or other form.

As a result of the invention, it is possible to take advantage of theoutstanding paste and serum viscosity of tomatoes which are homozygousfor the alc genes without sacrificing desirable tomato colorcharacteristics which are of importance to consumers. Also, the pasteand serum of the tomatoes enjoy excellent resistance to syneresis. It islikewise believed that homozygous rin tomatoes, homozygous nor tomatoes,homozygous Nr tomatoes, or heterozygous alc/rin, alc/nor, alc/Nr,rin/nor, Nr/nor, rin/Nr tomatoes can be advantageously used in thepresent invention.

The tomato paste according to the invention preferably has at 12° BrixBostwick thickness values in the range of from 0-3 cm, preferably from0-2 cm. Likewise preferred tomato pastes according to the inventionenjoy at 12° Brix syneresis levels of less than 4 mm, preferably lessthan 3 mm. This is in contrast to Bostwick values of 4.5-7 cm andsyneresis values of 13-25 mm for, e.g., the BOS 3155 variety(industry-known variety).

The invention may provide processed tomato products having both goodcolor and outstanding thickness, without requiring the mixing ofdifferent types of tomatoes. Preferably the pastes according to theinvention have a red (reddish), yellow (yellowish), orange (orangish),or pink (pinkish) color. More preferably, the pastes of the inventionhave a USDA color score at 8.5° Brix of at least 35, especially greaterthan 42. Preferably, said color scores are below 60. USDA scores arestandardised measurements for color quality.

We have found that it is possible to produce a tomato having bothhomozygous (what is believed to be) alc and the old gold crimson (ogc)genes, wherein the tomato color is good, yet at the same time tomatofruit firmness and juice and paste viscosity are excellent as a resultof the ripening inhibiting effect of the alc gene.

Following the above, the present invention relates to processed tomatoproducts such as tomato pastes, tomato sauces, tomato juices, tomatoconcentrates, tomato passatas, salsa, barbecue sauce, pizza sauce,spaghetti sauce, tomato fritto, ketchup (catsup), soup, pulp, dices(including products containing pulp and dices) and others, whichprocessed tomato products comprise tomatoes according to the invention.Preferably, the above products are prepared of tomatoes which furtherinclude color enhancing genes as well. Processed tomato productspreferably have a Brix value of 5-31°, preferably (depending upon theintended use) of 10-25°. Also depending upon the use they may contain0.1-5% wt, preferably 0.5-3% wt of salt, most preferably 1-2% wt. The pHmay suitably be between 3 and 5, preferably between 4.0 and 4.4.

Preferably, the invention concerns processed tomato products made frompopulations or assemblages of the above fruits having an average of atleast 10% by weight, and preferably at least 25%, more preferably atleast 50% of the tomatoes with the above-described genes. The tomatoesfor such processed tomato products may be obtained through classicalbreeding and selecting, but may also be obtained by geneticmodification, as is set out in WO 01/04315 and WO 01/14561.

The pastes of the invention preferably include at least 50% by weight ofthe tomatoes according to the invention, especially from 50 to 100% byweight. Juices preferably include at least 10% by weight of the tomatoesaccording to the invention, especially from 20 to 40% by weight.

Preferably, the tomatoes according to the invention are homozygous forthe color enhancing gene such as ogc, hp or dg.

Use of the tomatoes according to the invention is particularlybeneficial in view of their unique qualities, such as extremely highviscosity and almost no syneresis. It is believed these advantages arenot achieved with tomatoes or tomato pastes outside of our invention(when measured with equivalent soluble solids level and absence of otherthickening material, such as starch, gums, etcetera). A secondarybenefit is that as a result of such characteristics, less paste can beused in preparing a sauce. The advantageous paste characteristicsaccording to the invention can be expected to translate to improved,consumer perceivable characteristics for processed tomato products, suchas improved mouthfeel and texture and to lead to more full-bodied saucesand other products.

DETAILED DESCRIPTION OF THE INVENTION

Although it is believed tomatoes as such are known which are homozygousin rin, alc, or one or more other ripening-inhibiting genes mentioned,it is believed such tomatoes have never been used in tomato processing,and in fact it is reported that commercial use would not be suitable.Also, the tomatoes homozygous in rin, alc, or other genes that have beenstudied usually referred to tomatoes that do not form color. Hence,(industrially) processed tomato products (and tomato paste) having theproperties as described above are novel, and in particular suchprocessed tomato products that have a red or reddish color (e.g. USDAcolor score of at least 35, optionally less than 60). Furthermore, it isquite surprising that processed tomato products of good quality in termsof consistency and color could be achieved having the properties as nowclaimed, as tomatoes that are firm are usually associated with green,unripe tomatoes. Unripe, green tomatoes are unsuitable to use in largequantities in conventional tomato products, following the color and theflavour profile which is different from ripe tomatoes.

Without wishing to be bound by theory, it is believed that tomatoesaccording to the invention are different from conventional tomatoes inthat such gene mutations are not present in conventional tomatoes. Whensuch gene mutations are present in heterozygous, or more preferably inhomozygous form, they may interrupt part of the ripening process. It isbelieved that tomatoes according to the invention have different cellwalls, e.g. more dense cell walls.

It has been found that a tomato believed to be homozygous in alc haslevels of certain enzymes which are different from conventionaltomatoes. It was found that such a tomato, also comprising a color genesuch as ogc, had similar levels of exogalactanase when green as aconventional tomato. This is not surprising, but several days postbreaker (i.e. when pinkish/orange/red) the level of exogalactanasestayed low for (what was believed to be) an alc/ogc tomato, whereas forconventional tomato this level increases substantially. Regardingpolygalacturonase similar findings were obtained. Processing suchtomatoes into processed tomato products has distinct advantages. Still,thanks to the color gene ogc, such tomato has good color. Although aprocess may involve processing only such tomatoes, it may be preferredto use a blend of tomatoes: conventional tomatoes (for economic reasons)with tomatoes according to the invention. Preferably, such tomatoesaccording to the invention should also have one or more of the colorgenes as set out hereinbefore.

Hence, the present invention also relates to a process for preparing atomato product, the product being red (reddish), yellow (yellowish),orange (orangish), or pink (pinkish) and wherein at least 10% (pref. atleast 20%, more pref. at least 50%, up to 100%) of the tomatoes to beprocessed have a level of polygalacturonase of less than 200 (preferablyless than 100, more pref. less than 50, usually more than 1) μmolesGalA/ml/hour, and wherein said tomatoes to be processed have a level ofexogalactanase of less than 70 (preferably less than 50, most preferablyless than 35, usually more than 0.1) nmoles galactose/g fwt/hour(fwt=fresh weight). More preferably the product has a USDA color scoreof 35-60 and wherein at least 10% (pref. at least 20%, more pref. atleast 50%, up to 100%) of the tomatoes to be processed have a level ofpolygalacturonase of less than 200 (preferably less than 100, more pref.less than 50, usually more than 1) μmoles GalA/ml/hour, and wherein saidtomatoes to be processed have a level of exogalactanase of less than 70(preferably less than 50, most preferably less than 35, usually morethan 0.1) nmoles galactose/g fwt/hour (fwt=fresh weight). In this, it ispreferred that at least 10% (pref. 20%, more pref. 50%) of the tomatoesto be processed are homozygous for rin, homozygous for nor, homozygousfor Nr, homozygous for alc, heterozygous for combinations of two of therin, nor, Nr or alc genes, or combinations thereof. It may also bepreferred that the tomatoes are homozygous for at least two of rin, nor,Nr, or alc.

As (depending on amount used, and desired end product) it may bepreferred that the resulting product has some color, it is preferredthat the tomatoes as used in the process as set out above furthercomprise at least one color enhancing gene. For example, said colorenhancing genes may be selected from the group consisting of old goldcrimson (ogc), high pigment (hp), dark green (dg), intense pigment (Ip),as well as color enhancing transgenic genes.

The invention further relates to a tomato which was found by the presentinventors on which is believed to be alc, and has a specific 180 bpfragment following PCR amplification and Taq1 restriction of genomic DNAof said tomato (see example 5). Said tomato was following crossing withogc tomatoes ripening inhibited, but not green. Hence, the inventionfurther relates to red, orange, yellow or pink tomato showing a 180 bpfragment following PCR amplification and Taq1 restriction of genomic DNAof said tomato, as well as to a food product containing at least 10% wtof such tomato. It was found that such tomato was suitable for preparingpaste, tomato pulp, and tomato dices, and hence the invention alsorelates to a tomato paste, tomato pulp, tomato dices comprising at least10% wt (preferably at least 20% wt) of such tomatoes. The inventionfurther relates to processes as disclosed herein wherein the tomatoes tobe processed comprise at least 10%, preferably at least 20% wt of a red,orange, yellow or pink tomato showing a 180 bp fragment following PCRamplification and Taq1 restriction of genomic DNA of said tomato.

Conventional tomatoes are usually processed into paste using either acold-break process or a hot-break process. The hot-break processinvolves heating to above about 80° C. and comminuting (‘breaking’) thetomatoes, whereas a cold break would be heating to below about 80° C.and comminuting (‘breaking’) the tomatoes. The hot break process has asadvantage that endogenous enzymes are inactivated quickly, includingpectin-degrading enzymes like exogalactanase and polygalacturonase. Sucha product (e.g. paste) with substantial amount of long pectin-chains mayhave good consistency. The disadvantages are that heating may involvedamage to the flavour: a cooked or burnt aroma may develop, losingvolatiles and/or fruity flavours. The cold-break process does notde-activate the pectin-degrading enzymes quickly, so some degradation ofpectin may occur, resulting in a paste with less consistency. On theother hand, the flavour is usually better of a cold break product. Forthese reasons, mixtures of cold- and hot break products may be used.

Following the low level of pectin-degrading enzymes (polygalacturonaseand exogalactanase) of the tomatoes according to the invention, suchtomatoes may be processed using a so-called ‘cold break process’ andhave consistency more similar to hot break products, as it is believedby nature less pectin-degrading enzymes are present in the tomatoesaccording to the invention, and hence even if they are processed intopaste using a cold-break process such tomato paste may contain asubstantial amount of pectin. Thus, the invention also relates to aprocess comprising the steps of:

heating tomatoes to a temperature of 60-120° C. (preferably 60-80° C.),

size reduction (e.g. comminution) of said tomatoes

in any given order (optionally followed by concentration).

The tomato pastes and processed tomato products may be prepared usingconventional processing techniques.

As the tomatoes according to the invention are low in certain enzymes,the invention further relates to a process for preparing a tomatoproduct (e.g. paste or any other product) in which tomatoes are usedwhich are low in polygalacturonase and/or exogalactanase.

Hence, the invention further relates to a process for preparing a tomatoproduct, the product having a USDA color score of 35-60(i.e. red) andwherein at least 10% (pref. at least 20%, more pref. at least 50%, up to100%) of the tomatoes to be processed have a level of polygalacturonaseof less than 200 (preferably less than 100, more pref. less than 50,usually more than 1) μmoles GalA/ml/hour, and wherein said tomatoes tobe processed have a level of exogalactanase of less than 70 (preferablyless than 50, most preferably less than 35, usually more than 0.1)nmoles galactose/g fwt/hour.

In the above, it is preferred that at least 10% (pref. at least 20%,more pref. at least 50%, up to 100%) of the tomatoes to be processed arehomozygous for rin, homozygous for nor, homozygous for Nr, homozygousfor alc, heterozygous for combinations of two of the rin, nor, Nr or alcgenes, or combinations thereof.

The invention also relates to a process and product (i.e. tomato pasteand other processed tomato product) in which other ripening-inhibitinggenes than alc, rin, nor, Nr, are present in the tomato in such agenotypic form that they inhibit ripening similar to the tomatoes as areherein disclosed. This may relate to genes not yet known to inhibitripening, alone or in combination.

It is preferred that the tomatoes as used in the process as set outabove (e.g. to prepare a processed tomato product) further comprise atleast one color enhancing gene. For example, such color enhancing genesare selected from the group consisting of old gold crimson (ogc), highpigment (hp), dark green (dg), intense pigment (Ip), as well as colorenhancing transgenic genes.

The invention further relates to a process as set out above, wherein theprocess comprises the steps of:

heating tomatoes to a temperature of 60-120° C.,

comminuting or dicing said tomatoes

in any given order. More preferred in the above heating step is atemperature of 60-80° C. Optionally, a concentration step may beapplied, e.g. by water-removal, e.g. by evaporation.

EXAMPLES

In the experiments below, the following methods were followed.

Brix

A digital refractometer (Bellingham Stanley RFM 342 digitalrefractometer) thermostatically controlled at 20° C. was used. Therefractometer was calibrated with a range of 1-30% w/w sucrose inde-ionised water as standard solutions. Enough tomato product wasweighed into centrifuge tubes to provide a 1-2 ml liquid layer aftercentrifugation and centrifuged in a high speed centrifuge at 20° C.,using a Beckman Optima TLX ultracentrifuge (TLA100.4 8-position fixedangle rotor) having the following gradient: 5,000/2 min., 20,000/2 min.,75,000/4 min., 100,000/10 min., 50,000/1 min., end) at 95,000 RPM+/−5,000 RPM for 5 minutes to separate liquid from solid. Thecentrifugate liquid was placed in a small vial and mixed gently. Theliquid was placed on the optic of a thermostatted refractometer, the lidclosed and measured after the sample had sat for 30 seconds to reach therequired temperature. The average of triplicate readings was calculated.

Bostwick

Bostwick measurement was performed on a 25 cm Bostwick levelled in twodirections. Paste was diluted to 12° Brix and warmed or cooled to 20° C.Sample was placed in the Bostwick to the top of the sample chamber andthe trap door opened.

Degree of flow was determined after 30 seconds.

Samples were each tested in duplicate.

Insoluble Solids

Tomato paste was sourced from around the world including Unileverfactories in Chile (Malloa), California (Stockton/Merced), India andAustralia (Tatura) and external sources (Conesa, ARC, Copais). Thesewere used to construct a calibration line for tomato paste at 12 Brix.

A 1-1.5 g sample of juice was weighed out to 4 decimal places between 4pre-weighed filters (Whatman GFA 5.5 cm diameter). This was then placedon a Buchner vacuum filtration system and washed with 6 litres ofde-ionised water. The filters were then dried in a vacuum oven at 70° C.for 1.5 hours and then cooled in a dessicator to room temperature. Thefilters were then re-weighed and insoluble solids calculated as thefinal weight minus initial filter weight, divided by the initial juiceweight minus filter weight. Determination was carried out in triplicate.% insolubles=(weight of dried sample+filter)−filter/sample weight*100

Also, the dilution factor from paste to 5° Brix juice as above was takeninto account by separate multiplying.

Example 1 Breeding and Selection

As starting material a cross between a homozygous ripening-inhibitedmutant (thought to be alc; see example 5 for indentification) and ahomozygous old gold crimson (ogc) mutant was obtained from Ohio StateUniversity, USA (accession number 96-9422-400). This population of F1heterozygous alc/ogc was then selfed, and single plants selected thathad the phenotypical characteristics of both fruit ripening-inhibition(homozygous alc) and golden flower colour (homozygous ogc). Seed fromselected plants was then back-crossed with in-house breeding lines toproduce stable double homozygous plants, for evaluation of fruit andprocessed tomato product characteristics.

Example 2 Bostwick vs. Insoluble Solids: 22 Conventional Pastes(Control) and According to Invention

Of 22 hot and cold break tomato pastes from factories or commerciallyavailable the percentage of insoluble solids was measured, and theBostwick value was determined (all at 12° Brix). The Bostwick values(averages of various measurements per paste) were then plotted againstpercentage of insoluble solids.

The hot break pastes were either commercially available products, orwere prepared in own factories using the following process: the tomatoesare crushed with a minimum of air inclusion and quickly heated togreater than 85° C. typically through contact with a steam coil. Thejuice is then extracted and evaporated typically by a 2 or 3 stageprocess to between 24° and 31° Brix. For measuring, the samples werediluted to 12° Brix.

The cold break pastes were commercially available products, and aretypically prepared by crushing the tomatoes at temperatures of less than85° C. with a minimum of air inclusion. The juice is then extracted andevaporated by typically a 2 or 3 stage process to between 24 and 31°Brix. For measuring, the samples were diluted to 12° Brix.

The percentage insoluble solids was calculated as described earlier. TheBostwick value was determined using the method as described earlier. Theresults of these measurements are in table 1. TABLE 1 insoluble solidsand Bostwick of 22 commercially available pastes Insoluble solidsBostwick 1.756 7.6 2.0545 6.75 2.062 6.4 2.0845 6.6 2.162 6.3 2.217 5.82.353 5.8 2.368 5.5 2.413 5.5 2.568⁽¹⁾ 5.1 2.6515 5 2.674 4.9 2.678 4.22.7415 4.75 2.95⁽²⁾ 4.5 2.974 4 2.98⁽³⁾ 4.3 3.07 4.1 3.139 3.25 3.38 3.23.38 3.25 3.6⁽⁴⁾ 2.7Source of some of the pastes:⁽¹⁾Chilean Malloa⁽²⁾Unilever Van den Bergh's⁽³⁾CONESA⁽⁴⁾COPAIS

Two trial harvests of alc-ogc tomatoes (according to the invention) wereprocessed and Bostwick/insolubles measured in the same manner, withresults: Insoluble solids Bostwick 3.3 0.9 3.6 0.5

The results are set out graphically together with the results of table 1in FIG. 1. FIG. 1 also gives lines for the equations:(Bostwick value)=10.5−2.3822×(percentage of insoluble solids)  (1)(Bostwick value)=10.0−2.3822×(percentage of insoluble solids)  (2)(Bostwick value)=9.5−2.3822×(percentage of insoluble solids)  (3)

As can be seen, all conventional pastes tested have a Bostwick valuelarger than equation (1) above would give, at the insoluble solidsinterval of 2.5-3.6%.

Example 3 Exogalactanase Activity

Exogalactanase activity of four types of tomatoes were measured at threestages of maturity: in the green stage (i.e. well before breaker point),5-6 days post breaker, and 12-13 days post breaker. The four typestomatoes were: the tomato according to example 1, which is thought to behomozygous for alc and homozygous for ogc, Bos 3155 (as commerciallyavailable) U338 (internal breeding line with conventional ripening andcolor) and homozygous ogc (internal breeding line with conventionalripening).

The exogalactanase activity was expressed as nmoles galactose per gramfresh weight (fwt) per hour that could be converted. The galactoseconversion was measured using the following protocol.

Preparation of Extracts

Samples of tomato pericarp were taken from the frozen storage and placedin a 50 ml Falcon tube containing PVPP (1% w/v buffer). 1:1.5 (w:v) of0.2M NaPhosphate buffer pH7.5 was then added. This was left for 60 minsat 4° C. for the fruit to defrost slightly to allow a more uniformhomogenisation. A Polytron SEV was used for 1-2 mins to homogenise thefruit. The extract was stirred for 20 mins, left to stand for 20 minsthen centrifuged at 38,700×g for 20 mins (all performed at 4° C.). Thesupernatant was divided into 1 ml aliquots for the subsequent assays andfrozen at −20° C.

Exogalactanase Assay

Exogalactanase activity was measured by a linked assay consisting of twosteps. (1) Galactan was prepared as described [Methods in CarbohydrateChemistry Volume 5 (pp 132-134] and incubated with extract in thepresence of buffer. (2) D-galactose released in (1) was quantified byincubating with NAD and β-D-galactose dehydrogenase as described [Kurzand Wallenfels, 1974. Methods of enzymic analysis, 1279-1282. ed. VedagChemie, Weinheim].

1. The following components were mixed and incubated overnight at 30° C.(in duplicate): Test: 30 μl 10 mg/ml Lupin Galactan 15 μl 1M Na Acetate,pH 5.0 30 μl extract Control: 30 μl H₂O 15 μl 1M Na Acetate, pH 5.0 30μl extract Substrate Control: 30 μl 10 mg/ml Lupin Galactan 15 μl 1M NaAcetate, pH 5.0 30 μl H₂O

The reaction was stopped by incubating in a boiling water bath for 2mins.

2. To 64 μl of each incubated sample (above step 1) the following wereadded;

-   -   64 μl Galactose Dehydrogenase [2.5 U/ml]    -   905 μl 0.1 MTris/HCl pH8.6

O.D. readings were taken at 340 nm, following which 32 μl, 12.5 mg/mlNAD was added. This was incubated at room temperature for 1 hour and afurther reading at 340 nm was recorded (it was assumed that for eachmole of galactose released, one mole of NADH is formed). Test ΔOD₃₄₀(minus control and substrate control ΔODs) was converted to nmol gal/gfwt/hr using a galactose standard curve. The results are set out in FIG.2.

Example 4 Polygalacturonase Activity

Polygalacturonase activity of four types of tomato was measured at threestages of maturity: 5-6 days post breaker, and 12-13 days post breakerand 19-20 days post breaker. The four types of tomatoes as in example 3were analysed.

Polygalacturonase activity was measured using the PAHBAH method [Lever,M (1972) A new reaction for colorimetric determination of carbohydrates.Anal. Biochem. 47:273-279].

Materials: Solutions

Assay buffer stock: 50 mM sodium acetate buffer, pH 4.0 with 0.2M NaCl.

Substrate: Sigma polygalacturonic acid (PGA), 0.4% stock in water(fresh)

Standard: Sigma D-galacturonic acid, 50 mg/100 ml stock (fresh)

Enzyme extract: Tomato extracts were prepared as described in Example 3except that 2.5 volumes of buffer were used (in order to reduce thelevel of endogenous reducing sugars).

Megazyme fungal polygalacturonase (ammonium sulphate suspension) wasused as a positive control. Enzyme was diluted 1 in 1,000 in assaybuffer and stored at −20° C.

Prior to use the enzyme was diluted 1 in 25 in assay buffer stock(giving 1 in 25,000 final dilution) and 10-200 ul used per assay.

PAHBAH stock solution A: Slurry 10 g para-hydroxybenzoic acid hydrazide(Sigma) in 60 ml H₂O. Add 10 ml conc. HCl, mix and make up to 200 mlwith water (pale yellow solution, may be stored in fridge for severalweeks).

PAHBAH stock solution B: Dissolve 29.4 g trisodium citrate (0.05M) in500 ml water.

Add 2.2 g anhydrous (2.9 g dihydrate) calcium chloride (0.01M), mixwell. Add 40 g NaOH (0.5M), dissolve and make up to 2 litres with water(colourless solution, may be stored in fridge for several weeks).

Materials: Equipment

Boiling water bath or dry heating block.

Temperature controlled water bath (40° C.) or dry heating block.

UV spectrophotometer and cuvettes.

Teflon capped tubes (5 ml).

Method

1. 0.25 ml aliquots of 0.4% PGA were placed in teflon capped tubes.

2. 0.25 ml of enzyme extract was added (final concentrations are 0.2%PGA, in 25 mM Na Ac buffer pH 4.0 with 0.1M NaCl). A buffer onlynegative control, boiled diluted enzyme negative control and Megazyme PGpositive control were included at this stage. Tubes were centrifugedbriefly in a bench top microfuge (2,000 rpm) to ensure all the assaymixture was at the bottom of the tube. Assays were started in batches ofup to 24.

3. Assays were incubated at 40° C. for 1 hour (start of incubationequals T0).

4. After 1 hour incubation time, 5 ml of PAHBAH C (1 part of PAHBAH A to9 parts of PAHBAH B, mixed well and kept on ice) was added to each assayand immediately incubated at 100° C. for 6 minutes. For T0 controls, 5ml PAHBAH C was added to 0.25 ml of PGA, followed by 0.25 ml ofappropriate enzyme dilution.

5. Samples were cooled under running water.

6. A standard curve was made (in duplicate) using galacturonic acid(0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 ml of Gal A Stock (50 mg/100ml) per ml to give 59, 117, 235, 470, 705, 940 and 1175 nmoles GalA/0.5ml standards). 5 mls of PAHBAH C was added, incubated at 100° C. for 6minutes then cooled under running water. Gal A Stock (50 mg/100 ml) perml WATER per ml Nmoles Gal A/0.5 ml 0.5 0 1175 0.4 0.1 940 0.3 0.2 7050.2 0.3 470 0.1 0.4 235 0.05 0.45 117 0.025 0.475 59 0 0.5 07. OD readings were taken at 410 nm and the change in OD₄₁₀ over onehour (T60-T0) determined. These values were multiplied by an OD tonmoles galA conversion factor (from standard curve of OD₄₁₀ vs. nmolesGal A) and then by 10 (0.1 g tissue/assay extract) to give a final valueunit of nmoles galA produced/hour/g fresh weight. The results are setout in FIG. 3.

Example 5 Molecular Characterisation of Ripening-Inhibited Mutant

Definitive identification of ripening-inhibited mutants is difficult asmany accessions have been assigned to a particular nomenclature (e.g.nor, rin, Nr, alc) based on their ripening-inhibited phenotype.Recently, gene compositions involved in ripening-inhibition have beenreported for rin (WO01/14315) and nor (WO01/14561). Knowledge of thegene composition permits the development of a DNA-based molecular screenthat defines the particular ripening-inhibited mutant.

A ripening mutant (thought to be alc, and as used in the other examplesherein) is characterised by the fact that it yields fragments of approx393 bp, 180 bp and 35 bp following amplification of a region of genomicDNA using primers N12 and N13 and restriction of this amplificationproduct with Taq1. By contrast, normal ripening and someripening-inhibited mutants (e.g. nor) yield fragments of approx 393 bp,120 bp, 60 bp and 35 bp. The presence of the 180 bp fragment followingthis analysis therefore defines this ripening-inhibited fruit (thoughtto be alc). Interestingly, the 180 bp fragment is absent in the TomatoGenetic Resource Center (TGRC) alc accessions LA2529 and LA3134.Molecular screening for the presence of this 180 bp fragment was adescribed below.

Seeds from the ripening-inhibited mutant (used in the present examples,thought to be alc), TGRC alc accessions LA2529 and LA3134, inbred line108 (internal breeding line with conventional ripening and colour) andhybrid line 2010 (internal breeding line with conventional ripening andcolour) were germinated in compost (John Innes No 2) and maintained in aglasshouse (day/night temperatures 23° C./18° C.; 16 hr photoperiod).Leaf material was collected from plants ˜3 week post-germination andimmediately frozen in liquid nitrogen before placing at −80° C. prior toDNA extraction. Genomic DNA was extracted from ˜100 mgFwt frozen leafmaterial using QIAGEN DNeasy plant DNA extraction kit according tomanufacturers instructions with the inclusion of the optional 5 mincentrifugation step to remove cell and protein debris from the lysate.Genomic DNA was eluted from the QIAGEN column in 200 μl elution buffer(10 mM Tris.CL, pH8.0). Each DNA preparation was then further diluted(4-fold) prior to use as a target for PCR amplification.

For amplification, the oligonucleotide primers N12 and N13 weresynthesised by Sigma-Genosys and were provided lyophilised following adesalt purification. Both primers were resuspended in 10 mM Tris.ClpH7.5 to a final concentration of 100 pmol μl⁻¹.

The amplification reaction mix comprised 3 μl genomic DNA, 0.15 μlprimer N12 [5′-atcccaacatatcatgcaaatcatctat-3′], 0.15 μl primer N13[5′-taatgtactttaaccaggggcggctcta-3′], 15 μl JumpStart™ REDTaq™ ReadyMix™(Sigma-Aldrich), and 11.7 μl sterile distilled water. Reaction mixtureswere thermocycled at 94° C.-7 min, 35 cycles of [94° C.-45 sec, 53°C.-30 sec, 72° C.-90 sec] followed by a final extension step of 72° C.for 10 min. Following amplification, reaction products were restrictedby the addition of 2 μl Taq1 directly to the amplification reactionmixture and further incubation at 65° C. for 1 hour. Following Taq1restriction of amplification products, fragments were separated byelectrophoresis through a 1.5% (w/v) agarose gel and visualised usingethidium bromide and UV transillumination (see FIG. 4).

The photograph of the fragments following separation by agarose gelelectrophoresis clearly shows the presence of the 180 bp fragmentfollowing N12-N13 amplification and Taq1 restriction of genomic DNA fromthe ripening-inhibited mutant thought to be alc.

1. Tomato paste having an increased consistency such that when measuredat an insoluble solids interval of 2.5-3.6% at 12° Brix:(Bostwick value)<10.5−2.3822×(percentage of insoluble solids).
 2. Tomatopaste according to claim 1, such that when measured at insoluble solidsinterval of 2.5-3.6% at 12° Brix:(Bostwick value)<10.0−2.3822×(percentage of insoluble solids).
 3. Tomatopaste according to claim 2, such that when measured at an insolublesolids interval of 2.5-3.6% at 12° Brix:(Bostwick value)<9.5−2.3822×(percentage of insoluble solids).
 4. Tomatopaste according to claim 1, wherein the paste is obtained by a hot breakprocess, and optionally followed by concentration.
 5. Tomato pasteaccording to claim 1, having a red, yellow, pink, or orange color at8.5° Brix.
 6. Processed tomato product, comprising at least 10% (pref.20%, more pref. 50%) tomatoes which are homozygous for rin, homozygousfor nor, homozygous for Nr, homozygous for alc, heterozygous forcombinations of two of the rin, nor, Nr or alc genes, or combinationsthereof, whereby the term “processed tomato product” is defined as aproduct that comprises tomatoes which are subjected to a concentrationstep.
 7. Product according to claim 6 comprising tomatoes which arehomozygous for at least two genes of rin, nor, Nr, or alc.
 8. Productaccording to claim 6 having a red, yellow, pink, or orange color at 8.5Brix.
 9. Product according to claim 6 wherein said tomatoes furthercomprise at least one color-enhancing gene.
 10. Product according toclaim 9 wherein said color enhancing genes are selected from the groupconsisting of old gold crimson (ogc), high pigment (hp), dark green(dg), intense pigment (Ip), as well as color enhancing transgenic genes.11. Product according to claim 6, wherein the processed tomato productis in the form of tomato pastes, tomato sauces, tomato juices, tomatoconcentrates, tomato passatas, salsa, barbecue sauce, pizza sauce,spaghetti sauce, tomato fritto, ketchup (catsup), soup, pulp, dices orother form.
 12. Process for preparing a tomato product, the producthaving a red, yellow, pink, or orange color and wherein at least 10%(pref. 20%, more pref. 50%) of the tomatoes to be processed have a levelof polygalacturonase of less than 200 (preferably less than 100, morepref. less than 50) μmoles GalA/ml/hour, and wherein said tomatoes to beprocessed have a level of exogalactanase of less than 70 (preferablyless than 35) nmoles galactose/g fwt/hour.
 13. Process according toclaim 12, wherein at least 10% (pref. 20%, more pref. 50%) of thetomatoes to be processed are homozygous for rin, homozygous for nor,homozygous for Nr, homozygous for alc, heterozygous for combinations oftwo of the rin, nor, Nr or alc genes, or combinations thereof. 14.Process according to claim 12, wherein other ripening-inhibiting genesthan alc, rin, nor, Nr, are present in the tomato in such a genotypicform that they inhibit ripening.
 15. Process according to claim 12,wherein said tomatoes further comprise at least one color enhancinggene.
 16. Process according to claim 15, wherein said color enhancinggenes are selected from the group consisting of old gold crimson (ogc),high pigment (hp), dark green (dg), intense pigment (Ip), as well ascolor enhancing transgenic genes.
 17. Process according to claim 12,wherein the process comprises the steps of: heating tomatoes to atemperature of 60-120° C. comminuting or dicing said tomatoes in anygiven order.
 18. Red, orange, yellow or pink tomato showing a 180 bpfragment following PCR amplification using oligonucleotides5′-atcccaacatatcatgcaaatcatctat-3′ and5′-taatgtactttaaccaggggcggctcta-3′ and Taq1 restriction of genomic DNAof said tomato.
 19. Food product containing at least 10% wt of thetomato according to claim
 18. 20. Tomato paste, tomato pulp, tomatodices comprising at least 10% wt of the tomato according to claim 18.21. Process according to claim 12, wherein the tomatoes comprise atleast 10% wt of tomatoes.